An adsorbent for removing protein-bound and medium-large molecular weight toxins and a method for preparing the same

Abstract

The application discloses an adsorbent for removing protein-bound toxins and middle-molecular toxins and a preparation method thereof, and belongs to the technical field of polymer materials, and comprises the following steps: polystyrene-based white ball preparation, amine immobilization and film coating.The application has the beneficial effects that: a kind of adsorbent capable of adsorbing protein-bound toxins and middle-molecular toxins is prepared by adding a pore-forming agent under the participation of styrene monomers, polyvinyl benzene series, glycidyl methacrylate and 4,4'-bis(methacrylamido)-azobenzene, the adsorption effect is good, and the adsorbent has high selectivity; the process method is simple, green and environment-friendly; glycidyl methacrylate and modified cyclodextrin are added, the hydrophilicity of the adsorbent is enhanced, the blood compatibility of the adsorbent is improved, and the adsorption effect of the adsorbent is enhanced through amine group immobilization.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of high polymer material, more particularly relates to an adsorbent for removing protein-bound toxins and middle-molecular weight toxins and a preparation method thereof. BACKGROUND

[0002] Chronic kidney disease (CKD) is a chronic disease caused by damage to kidney function due to diabetes, hypertension, and various primary kidney diseases. At present, CKD is a public health problem endangering human health worldwide. Patients with CKD stage 5 have a high cardiovascular morbidity and mortality. Cardiovascular disease (CVD) is a common complication and the leading cause of death in CKD patients. Uremic toxins are a specific risk factor for CVD in CKD patients.

[0003] Studies have shown that uremia can cause complications such as water and electrolyte and acid-base metabolism disorders, cardiovascular disease, nervous system disease, and skin itching, which seriously affect the physical and mental health and life safety of patients. It is currently known that the concentration of about 200 substances in uremic patients is higher than that in normal people, mainly including three types of small molecule toxins, middle-molecular weight toxins, and protein-bound toxins. Small water-soluble solute molecules have a molecular weight of less than 500 Da, such as creatinine and urea. Middle-molecular weight solutes have a molecular weight of more than or equal to 500 Da, and the representative solute is beta2-microglobulin and parathyroid hormone. Protein-bound toxins represented by p-cresol sulfate (PCS) and indoxyl sulfate (IS) account for 24% of uremic toxins and can bind to serum albumin to form protein-bound uremic toxins. A large number of studies have confirmed that PCS and IS are positively correlated with all-cause mortality in CKD patients.

[0004] Blood purification, as the main treatment for patients with end-stage renal disease, plays an important role in removing uremic toxins. However, the preparation process of the adsorbent in the existing blood purification system is complicated, and the adsorption selectivity of middle-molecular weight toxins and protein-bound toxins is poor and cannot be regenerated.

[0005] Shape memory polymer refers to a new type of functional polymer material that can change its physical or chemical properties in response to external stimuli to adapt to the surrounding environment. External stimuli include light, temperature, pH, electromagnetic field, etc. Azobenzene and its derivatives are common shape memory polymers that can undergo trans to cis transformation under ultraviolet light irradiation to realize the regeneration of the adsorbent resin.

[0006] The current shape memory polymer polymer can be regenerated as a protein-bound toxin and macromolecular toxin adsorbent, but the existing shape memory polymer has good regenerability, but has problems of poor adsorption selectivity and poor blood compatibility, in order to solve the above problems, the application prepares a kind of protein-bound toxin and macromolecular toxin adsorbent and its preparation method. SUMMARY

[0007] In order to solve the above problems, overcome the shortcomings of the prior art, the application provides a kind of protein-bound toxin and macromolecular toxin adsorbent and its preparation method to solve the existing adsorbent preparation process is complicated, adsorption selectivity is poor and blood compatibility is poor.

[0008] The specific technical scheme for solving the above technical problems is: the preparation method of the protein-bound toxin and macromolecular toxin adsorbent, comprising the following steps:

[0009] (1) Preparation of polystyrene-based white ball:

[0010] The styrene monomer, multi-vinyl benzene, glycidyl methacrylate, 4,4'-bis (methacrylamide) -azobenzene, pore-forming agent and initiator are mixed as oil phase, dispersant, inorganic salt and pure water are mixed as water phase, two phases are mixed, and suspension polymerization is carried out under certain temperature and stirring rate, polystyrene-based white ball is obtained, and then it is washed, extracted and dried;

[0011] (2) amination immobilization;

[0012] The polystyrene-based white ball obtained in step (1) is added into an amination agent for amination treatment, and then the amination polystyrene resin is obtained after washing;

[0013] (3) film coating:

[0014] The amination polystyrene resin is dissolved in N,N-dimethylformamide (DMF) solution, sodium carbonate is added, modified dextrin 6-OTs-β-CD is dissolved by stirring, N2 is introduced, the temperature is increased to a certain time, and then the adsorbent is obtained after filtration, DMF-anhydrous ethanol precipitation and water washing;

[0015] As preferred, the styrene monomer is one or more of styrene, methylstyrene, ethylstyrene or 4-vinyl biphenyl; the multi-vinyl benzene is one or more of divinylbenzene, divinyl ethyl benzene and triallyl isocyanurate,

[0016] As preferred, the pore-forming agent is one or a mixture of two to three of toluene, xylene, chlorobenzene, 3-12 carbon atom alcohol, 5-12 carbon atom alkane, 200# gasoline or liquid paraffin, and the addition amount of the pore-forming agent is 50-300%.

[0017] As a preferred, the initiator is one or more of azobisisobutyronitrile, azobisisoheptyl nitrile, benzoyl peroxide, tert-butyl peroxy-2-ethylhexanoate, tert-amyl peroxy-2-ethylhexanoate, dodecanoyl peroxide or alkyl hydroperoxide;

[0018] The amount of the initiator is 0.5%-5% of the sum of the mass of the styrene monomer, the polyvinylbenzene, the glycidyl methacrylate and the 4,4'-bis(methacrylamido)-azobenzene;

[0019] As a preferred, the dispersant is a water-soluble polymer, and the content is 0.1-8% of the mass of the water phase;

[0020] The water-soluble polymer is one or two of polyvinyl alcohol, polyvinylpyrrolidone, gelatin, polyethylene glycol, carboxymethyl cellulose or hydroxyethyl cellulose;

[0021] As a preferred, in the step (1), the mass ratio of the oil phase to the water phase is 1:1-1:5; the reaction time is 10-20h; and the reaction temperature is 55-90℃;

[0022] Especially, the preparation method of the modified dextrin 6-OTs-β-CD is as follows:

[0023] After the cyclodextrin (β-CD) is added into water, sodium hydroxide is dissolved in water and slowly added into the flask, under the condition of ice water bath, p-toluenesulfonyl chloride dissolved in acetonitrile is slowly added into the reaction solution, after the reaction, the pH is adjusted, and after the filtration and recrystallization twice, the modified cyclodextrin 6-OTs-β-CD is obtained by vacuum drying;

[0024] Especially, in the step (1), the preparation method of the 4,4'-bis(methacrylamido)-azobenzene is as follows:

[0025] In pyridine, 4,4'-diaminoazobenzene solution is added and stirred at room temperature, methacryloyl chloride is gradually added, after the complete addition, the reaction mixture is heated for a period of time, after the cooling, the mixture is poured into ice, acidified with hydrochloric acid, filtered, washed with saturated sodium bicarbonate solution, then washed with water, and the crude product is recrystallized by using ethanol;

[0026] Especially, the aminating agent is at least one of ethylenediamine, hexanediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, polyethyleneimine and polylysine;

[0027] An adsorbent for removing protein-bound toxins and medium-molecular-weight toxins, which is prepared by the preparation method of the adsorbent.

[0028] The beneficial effects of the present application are as follows:

[0029] This invention utilizes styrene monomers and polyvinylbenzene compounds, with the participation of glycidyl methacrylate and 4,4'-bis(methacrylamido)-azobenzene, to prepare an adsorbent capable of adsorbing protein-bound toxins and medium- to large-molecule toxins by adding a porogen. The adsorption effect is good and has high selectivity. The process of this invention is simple, green and environmentally friendly.

[0030] This invention creatively incorporates glycidyl methacrylate and modified cyclodextrin, which enhances the hydrophilicity of the adsorbent and improves its blood compatibility.

[0031] This invention creatively enhances the adsorption effect of the adsorbent by immobilizing amine groups. Attached Figure Description

[0032] Appendix Figure 1 These are hemolysis experiment diagrams related to the embodiments and comparative examples in this invention;

[0033] Appendix Figure 2 These are platelet count diagrams from the embodiments and comparative examples of this invention;

[0034] Appendix Figure 3 These are coagulation experiment diagrams related to the embodiments and comparative examples in this invention;

[0035] Appendix Figure 4 This is a diagram illustrating the preparation of polystyrene white spheres in this invention;

[0036] Appendix Figure 5 This is a structural diagram of the resin microspheres after amino immobilization in this invention;

[0037] Appendix Figure 6 This is a structural diagram of the coated resin microspheres in this invention; Detailed Implementation

[0038] Specific details in the description of this invention are merely to provide a thorough understanding of the embodiments thereof; however, those skilled in the art should understand that the implementation of this invention is not limited to these details. Furthermore, well-known structures and functions have not been described or shown in detail to avoid obscuring the key points of the embodiments of this invention. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0039] Specific embodiments of the present invention:

[0040] To better understand the present invention, specific embodiments are described. It is worth emphasizing that the effects of these embodiments are not substantially different from those of various embodiments within the scope of protection of the present invention, including their respective reagents and reagent content ratios. All of them can achieve the effects described in the present invention and solve the above-mentioned problems. Other combinations are not described here. Example 1

[0041] (1) Preparation of 4,4'-bis(methacrylamido)-azobenzene:

[0042] A solution of 4 g of 4,4'-diaminoazobenzene was added to 40 ml of pyridine and stirred at room temperature, and 7 ml of methacryloyl chloride was gradually added. After complete addition, the reaction mixture was heated to 60°C for 1.5 h. After cooling, the mixture was poured into ice and acidified to pH 4 with hydrochloric acid, filtered, washed with saturated sodium bicarbonate solution, then with water, and the crude product was recrystallized from ethanol;

[0043] (2) Preparation of polystyrene-based white balls

[0044] In a beaker, 18 g of styrene, 6 g of divinylbenzene, 0.45 g of dodecanoyl peroxide, 6 g of glycidyl methacrylate, 2 g of 4,4'-bis(methacrylamido)-azobenzene, 12 g of n-heptane, and 20 g of toluene were mixed to form an oil phase. The mixture was stirred until the solids were completely dissolved. A water phase of 110 g was prepared, containing 1.5% PVA and 0.5% NaCl. The oil phase was dispersed in the water phase under stirring at a speed of 120 rpm. The temperature was raised to 55-60°C to start the polymerization, and the reaction was continued for 5 h at 85-90°C. The product was washed with hot water and ethanol several times, and then extracted with ethanol for 6 h to remove the oligomers. The product was dried under vacuum for 5 h and stored for future use.

[0045] (3) Immobilization of tetraethylenepentamine

[0046] 50 ml of polystyrene-based white balls were added to a 500 ml three-necked flask, and 200 ml of an aqueous solution was added. Tetraethylenepentamine was added, and the reaction was completed after mechanical stirring at 60°C for 12 h. The product was washed with water to obtain a grafted tetraethylenepentamine polystyrene resin;

[0047] (4) Encapsulation

[0048] 6 g of β-CD was weighed into a 50 ml round-bottom flask, and 25 mL of water was added. Then, 0.68 g of sodium hydroxide was dissolved in 2 mL of water and slowly added to the flask. Under ice water bath conditions, 1.5 g of p-toluenesulfonyl chloride dissolved in 3 mL of acetonitrile was slowly added to the reaction solution. After 2.5 h of reaction, the pH was adjusted to 6, and the product was filtered and recrystallized twice in hot water at 85°C. Finally, the product was dried under vacuum at 60°C to obtain 6-OTs-β-CD with a p-toluenesulfonyl group;

[0049] Take the resulting tetraethylene five amine polystyrene resin 10 ml in step (3) in 30 ml DMF solution, add 0.5 g of sodium carbonate, 5 g of 6-OTs-β-CD stirring and dissolving, N2, heating to 60-65℃ reaction 18 h, filter, DMF-anhydrous ethanol precipitation after water washing, get the target adsorbent. Example 2

[0050] (1) 4,4'-bis(methacrylamido)-azobenzene preparation:

[0051] In 100 ml of pyridine, add 10 g of 4,4'-diaminoazobenzene solution and stir at room temperature, gradually add 17.5 ml of methacryloyl chloride, after complete addition, heat the reaction mixture to 60℃ for 1 h, after cooling, pour the mixture into ice, acidify with hydrochloric acid to pH 4, filter, wash with saturated sodium bicarbonate solution, then wash with water, and recrystallize the crude product from ethanol;

[0052] (2) Preparation of polystyrene-based white ball

[0053] In a beaker, add styrene (6 g), divinylbenzene (18 g), benzoyl peroxide (1 g), glycidyl methacrylate (7 g), 4,4'-bis(methacrylamido)-azobenzene (6 g), n-butanol (30 g), and toluene (40 g) to form an oil phase. Stir until the solids are completely dissolved. Prepare an aqueous phase of 315 g (containing 2% PVP and 3% NaCl). Under stirring conditions, disperse the oil phase in the aqueous phase to form an O / W emulsion. The stirring speed is 60 rpm. Increase the temperature to 70-75℃ to start the polymerization. Polymerize for 5 h, then increase the temperature to 80℃ and continue to react for 12 h. Wash with hot water and ethanol several times, then extract with ethanol for 6 h to remove the oligomers inside the microspheres. Vacuum dry for 5 h and reserve.

[0054] (3) Immobilization of polyethyleneimine

[0055] Take 50 ml of polystyrene-based white ball beads and add them to a 500 ml three-necked flask. Add 200 ml of aqueous solution and add polyethyleneimine. Stir mechanically at 60℃ for 12 h to complete the reaction. Wash with water to obtain grafted ethyleneimine polystyrene resin.

[0056] (4) Film coating

[0057] Weigh 12 g of β-CD in a 50 ml round-bottom flask. Add 50 mL of water, then slowly add 1.36 g of sodium hydroxide dissolved in 4 mL of water to the flask. Under ice water bath conditions, slowly add 3 g of p-toluenesulfonyl chloride dissolved in 6 mL of acetonitrile to the reaction solution. After 2.5 h of reaction, adjust the pH to 6, filter, recrystallize twice in hot water at 85℃, and vacuum dry at 60℃ to obtain 6-OTs-β-CD with p-toluenesulfonyl group.

[0058] Take the ethylene imine polystyrene resin 10 ml obtained in step (3) and dissolve it in 30 ml of DMF solution, add 0.5 g of sodium carbonate, 5 g of 6-OTs-β-CD, stir and dissolve, pass N2, and heat to 60°C for 24 h. After filtration, DMF-anhydrous ethanol precipitation and water washing, the target adsorbent is obtained; Example 3

[0059] (1) Preparation of 4,4'-bis(methacrylamido)-azobenzene:

[0060] Add 20 g of 4,4'-diaminoazobenzene solution in 200 ml of pyridine and stir at room temperature. Gradually add 35 ml of methacryloyl chloride. After complete addition, heat the reaction mixture to 60°C for 1 h. After cooling, pour the mixture into ice and acidify with hydrochloric acid to pH 4. Filter, wash with saturated sodium bicarbonate solution, and then with water. Recrystallize the crude product from ethanol;

[0061] (2) Preparation of polystyrene-based white balls

[0062] In a beaker, add styrene (12 g), divinylbenzene (12 g), azobisisobutyronitrile (2 g), glycidyl methacrylate (6 g), 4,4'-bis(methacrylamido)-azobenzene (10 g), liquid paraffin (80 g), and xylene (30 g) to make an oil phase. Stir until the solids are completely dissolved. Prepare an aqueous phase of 720 g (containing 3% gelatin and 5% NaCl). Under stirring, disperse the oil phase in the aqueous phase to make an O / W emulsion. The stirring speed is 100 rpm. Raise the temperature to 70°C to start the polymerization. Continue the polymerization for 6 h at 85-90°C. Wash with hot water and ethanol several times. Then extract with ethanol for 6 h to remove the oligomers inside the microspheres. Vacuum dry for 5 h to obtain the product;

[0063] (3) Immobilization of ethylenediamine

[0064] Take 50 ml of polystyrene-based white ball beads and add them to a 500 ml three-necked flask. Add 200 ml of aqueous solution and add ethylenediamine. Stir mechanically at 60°C for 12 h to complete the reaction. Wash with water to obtain the grafted ethylenediamine polystyrene resin;

[0065] (4) Coating

[0066] Take 24 g of β-CD in a 50 ml round-bottom flask, add 100 ml of water, then slowly add 2.72 g of sodium hydroxide dissolved in 8 ml of water into the flask, and slowly add 6 g of p-toluenesulfonyl chloride dissolved in 12 ml of acetonitrile into the reaction solution under ice water bath conditions. After 2.5 h of reaction, adjust the pH to 6, filter, recrystallize twice in hot water at 85°C, and dry at 60°C under vacuum to obtain 6-OTs-β-CD with a p-toluenesulfonyl group;

[0067] Take 24 g of β-CD in a 50 ml round-bottom flask, add 100 ml of water, then slowly add 2.72 g of sodium hydroxide dissolved in 8 ml of water into the flask, and slowly add 6 g of p-toluenesulfonyl chloride dissolved in 12 ml of acetonitrile into the reaction solution under ice water bath conditions. After 2.5 h of reaction, adjust the pH to 6, filter, recrystallize twice in hot water at 85°C, and dry at 60°C under vacuum to obtain 6-OTs-β-CD with a p-toluenesulfonyl group;

[0068] In order to more intuitively show the process advantages of the present application, the preparation method of the protein-bound toxin and macromolecular toxin adsorbent of the present application and the same process are compared by using equivalent replacement method,

[0069] Comparative Example 1

[0070] In the preparation of polystyrene-based white balls, no modified cyclodextrin is added, and the remaining steps are the same as those of Example 2.

[0071] Comparative Example 2

[0072] In the preparation of polystyrene-based white balls, cyclodextrin is added, and the remaining steps are the same as those of Example 2; the decrease in adsorption performance may be due to the blockage of the pore structure;

[0073] Comparative Example 3

[0074] In the preparation of polystyrene-based white balls, the modified cyclodextrin of the reference is added, and the remaining steps are the same as those of Example 2; the modified cyclodextrin is prepared by referring to the preparation method of the modified cyclodextrin disclosed in 202110645891.6 "Vinyl monomer-polyvinyl crosslinking agent copolymer non-porous microsphere and its preparation method and application",

[0075] Comparative Example 4

[0076] In the preparation of polystyrene-based white balls, glycidyl methacrylate is replaced by epichlorohydrin;

[0077] Comparative Example 5

[0078] The preparation method of polystyrene-based white balls is the same as that of Example 2, and the second step of immobilizing amino groups is not performed.

[0079] Comparative Example 6

[0080] In the preparation of polystyrene-based white balls, the amine reagent is replaced by octanediamine.

[0081] Comparative Example 7

[0082] The polystyrene-based white ball was prepared according to the method of Example 2, except that no pore-forming agent was added;

[0083] Comparative Example 8

[0084] The polystyrene-based white ball was prepared according to the method of Example 2, except that the pore-forming agent was ethyl acetate;

[0085] The specific operation method for evaluating the adsorption performance is as follows:

[0086] A plurality of 10 mL samples containing 40 mg / L of indoxyl sulfate and p-cresol sulfate were taken, 1 mL of the adsorbent obtained in the examples and comparative examples was added, and after oscillation at 37°C for 2 h, the adsorption effect of the adsorbent on indoxyl sulfate and p-cresol sulfate was tested by high performance liquid chromatography, and the test results are shown in Table 2.

[0087] The adsorption performance of the adsorbent on parathyroid hormone was evaluated using the ELISA method, and the adsorption performance of the adsorbent on β2-microglobulin was evaluated using the immunoturbidity method, and the test results are shown in Table 2.

[0088] Table 1: Comparison of differences in adsorption and biocompatibility of different examples and comparative examples:

[0089] Experiment No. Group Glycidyl methacrylate Modified cyclodextrin Immobilized amine reagent of amino group Pore former 1# Example 1 + + + + 2# Example 2 + + + + 3# Example 3 + + + + 4# Comparative Example 1 + None + + 5# Comparative Example 2 + Cyclodextrin + + 6# Comparative Example 3 + Modified cyclodextrin + + 7# Comparative Example 4 Epichlorohydrin + + + 8# Comparative Example 5 + + None + 9# Comparative Example 6 + + Octanediamine + 10# Comparative Example 7 + + + None 11# Comparative Example 8 + + + Ethyl acetate

[0090] The results are shown in Table 2 below:

[0091] Table 2: Clearance effect of adsorbent on protein-bound toxins and macromolecular toxins

[0092] Experiment No. Sample Indoxyl sulfate Sulfate to cresol Beta 2-microglobulin Parathyroid hormone 1# Example 1 80% 78% 85% 97% 2# Example 2 85% 88% 91% 92% 3# Example 3 83% 85% 88% 87% 4# Comparative Example 1 57% 62% 80% 85% 5# Comparative Example 2 55% 49% 73% 72% 6# Comparative Example 3 58% 60% 82% 84% 7# Comparative Example 4 50% 52% 75% 74% 8# Comparative Example 5 55% 49% 73% 72% 9# Comparative Example 6 63% 70% 72% 80% 10# Comparative Example 7 53% 59% 50% 48% 11# Comparative Example 8 62% 55% 69% 70%

[0093] (1) Example 2 and Comparative Examples 1-3 are an analysis of the influence of the technical feature of "modified cyclodextrin" on the adsorption effect of the present application,

[0094] Among them, Comparative Example 1 does not add modified cyclodextrin; Comparative Example 2 adds ordinary cyclodextrin, and Comparative Example 3 adds modified cyclodextrin according to the reference, and through comparison of experimental data,

[0095] Comparative Example 2 and Comparative Example 3, cyclodextrin does not participate in the reaction, but the adsorption effect of p-cresol sulfate, β2-microglobulin and parathyroid hormone in Comparative Example 2 is significantly decreased, which may be that the water solubility of cyclodextrin itself is poor and it is difficult to be washed out and block the pore, while the modified cyclodextrin in Comparative Example 3 has significantly enhanced hydrophilicity and is easily washed out, resulting in almost no change in adsorption performance compared with Comparative Example 1 without adding cyclodextrin;

[0096] Comparative Examples 1-3, the adsorption effect of the adsorbent on indoxyl sulfate, p-toluenesulfonic acid, β2-microglobulin and parathyroid hormone is far lower than the adsorption effect of the present application. Therefore, the double bond modified modified cyclodextrin with p-toluenesulfonyl prepared by the present application has a positive effect on the adsorption effect of indoxyl sulfate, p-toluenesulfonic acid, β2-microglobulin and parathyroid hormone compared with not adding, adding ordinary cyclodextrin, and adding other double bond modified modified cyclodextrin.

[0097] (2) Example 2 and Comparative Example 4 are an analysis of the influence of the "ring-opening reagent" technical feature on the creativeness of the adsorption effect of the present application.

[0098] Among them, glycidyl methacrylate as a ring-opening reagent can enhance the hydrophilicity of the adsorbent, which is beneficial to improve the blood compatibility of the adsorbent. However, epichlorohydrin as a ring-opening reagent cannot participate in the reaction, and the decrease in hydrophilicity leads to lower adsorption effect, especially the adsorption effect of indoxyl sulfate and p-toluenesulfonic acid is only 57-60%.

[0099] (3) Example 2 and Comparative Examples 5-6 are an analysis of the influence of the "amine group immobilization" technical feature on the creativeness of the adsorption effect of the present application.

[0100] Comparative Example 5 is different in that the second step of amino group immobilization is not performed. Although Comparative Example 6 performs amino group immobilization, the amination reagent is replaced by octanediamine. Through comparison of experimental data, it can be seen that Comparative Examples 5-6 have lower adsorption effect compared with the present application. Therefore, the immobilization of amino groups, especially the immobilization of polyethyleneimine amino groups, has a positive promoting effect on the adsorption effect in the present application.

[0101] (4) Example 2 and Comparative Examples 7-8 are an analysis of the influence of the "porogen" technical feature on the creativeness of the adsorption effect of the present application.

[0102] Comparative Example 7 does not add a porogen, while Comparative Example 8 uses ethyl acetate as a porogen. Through comparison of experimental data, it can be seen that the addition of porogen, especially toluene and n-butanol, has a positive promoting effect on the adsorption effect compared with ethyl acetate.

[0103] It is worth noting that, compared with Comparative Examples 7-8, although Comparative Example 8 adds a porogen of ethyl acetate, the adsorption effect on indoxyl sulfate, β2-microglobulin and parathyroid hormone has been improved to a certain extent, but the adsorption capacity on p-toluenesulfonic acid is inversely affected compared with not adding a porogen, decreasing from 59% to 55%. Therefore, the addition of porogen does not have a complete positive correlation with the adsorption capacity.

[0104] In summary, the factors of "modified cyclodextrin", "immobilization of amino groups" and "porogen" have a positive promoting effect on the adsorption effect of the present application.

[0105] In order to more intuitively show the process advantages of the present application, further research and investigation on biocompatibility were carried out, wherein

[0106] The biocompatibility test results are as follows:

[0107] The hemolysis test method refers to YY / T1651.1-2019 Medical Devices - Hemolysis Test Part 1: Material-mediated Hemolysis Test ( Figure 3 );

[0108] The hematology test method refers to YY / T1649.1-2019 Medical Devices and Platelet Interaction Test Part 1: In vitro Platelet Counting Method ( Figure 4 );

[0109] Coagulation YY / T1911-2023 Medical Devices Coagulation Test Method ( Figure 5 )

[0110] The above blood compatibility test results refer to Table 3:

[0111] Table 3 Blood Compatibility Test Data

[0112] Experiment No. Sample Hemolysis rate Platelet adhesion rate Coagulation time ratio 1# Example 1 1.3% 4.2% 98.7% 2# Example 2 0.9% 7.1% 95.4% 3# Example 3 0.5% 6.2% 93.6% 4# Comparative Example 1 2.1% 15.3% 81.2% 5# Comparative Example 2 1.8% 14.7% 84.3% 6# Comparative Example 3 2.2% 15.9% 80.7% 7# Comparative Example 4 4.8% 17.8% 78.0% 8# Comparative Example 5 2.2% 13.6% 75.9% 9# Comparative Example 6 3.4% 18.4% 83.0% 10# Comparative Example 7 3.3% 15.2% 81.2% 11# Comparative Example 8 2.7% 22.1% 82.3% Index Less than 5% More than 80%

[0113] (1) Example 2 and Comparative Examples 1-3 are for analyzing the creative influence of the "modified cyclodextrin" technical feature on the biocompatibility of the present application,

[0114] Among them, Comparative Example 1 does not add modified cyclodextrin; Comparative Example 2 adds ordinary cyclodextrin, and Comparative Example 3 adds the modified cyclodextrin of the reference. Through comparison of experimental data, the blood compatibility of the adsorbent is investigated, and the hemolysis rate, platelet adhesion rate and coagulation time ratio are compared. Although the present application is superior to Comparative Examples 1-3 in terms of hemolysis rate, platelet adhesion rate and coagulation time indicators, the indicators of Comparative Examples 1-3 also basically meet the hemolysis rate less than 5% and the coagulation time ratio greater than 80%. It can be seen that the "modified cyclodextrin" technical feature has a certain influence on the biocompatibility of the present application, but it is not the dominant factor.

[0115] (2) Example 2 and Comparative Example 4 are for analyzing the influence of the technical feature of "ring-opening reagent" on the biocompatibility of the present application. GMA as a ring-opening reagent can enhance the hydrophilicity of the adsorbent, which is beneficial to improving the blood compatibility of the adsorbent. However, compared with epichlorohydrin as a ring-opening reagent, the hemolysis rate, platelet adhesion rate and blood clotting time ratio of the blood compatibility are close to the critical value of 5%, especially the blood clotting time ratio is lower than 80%, which does not meet the requirements.

[0116] Therefore, GMA as a ring-opening reagent has a significant influence on the blood clotting time ratio of the biocompatibility of the present application.

[0117] (3) Example 2 and Comparative Examples 5-6 are for analyzing the influence of the technical feature of "amino group immobilization" on the biocompatibility of the present application.

[0118] Comparative Example 5 is different in that "the second step of amino group immobilization is not performed". Although Comparative Example 6 performs amino group immobilization, the aminating reagent is replaced by octanediamine. According to the comparison of experimental data, the hemolysis rate of the present application is better than that of Comparative Examples 5-6, and overall, it meets the requirement of less than 5%. However, the blood clotting time ratio of Comparative Examples 5-6 is lower than 80%, which does not meet the requirements.

[0119] Therefore, the technical feature of "amino group immobilization" has a significant influence on the blood clotting time ratio of the biocompatibility of the present application.

[0120] (4) Example 2 and Comparative Examples 7-8 are for analyzing the influence of the technical feature of "porogen" on the biocompatibility of the present application.

[0121] Comparative Example 7 does not add a porogen, while Comparative Example 8 uses ethyl acetate as a porogen. According to the comparison of experimental data, the performance of the present application is better than that of Comparative Examples 7-8. Overall, the hemolysis rate meets the requirement of less than 5%, and the blood clotting time ratio is basically greater than 80%. Therefore, the technical feature of "porogen" has a certain influence on the biocompatibility of the present application, but it is not the dominant factor.

[0122] Therefore, the factors of "modified cyclodextrin", "amino group immobilization" and "porogen" have a positive promoting effect on the biocompatibility of the present application. Among them, GMA as a ring-opening reagent and "amino group immobilization" have a significant promoting effect on the biocompatibility.

[0123] In summary:

[0124] (1) The adsorbent and the preparation method can simultaneously adsorb protein-bound toxins and medium and large molecule toxins, have good adsorption effect and high selectivity compared with traditional adsorbents, glycidyl methacrylate and cyclodextrin components are added in the reaction process, the hydrophilicity of the adsorbent is enhanced, and the blood compatibility of the adsorbent is improved; in addition, the process method is simple, green and environment-friendly;

[0125] (2) The adsorbent prepared by creatively adding glycidyl methacrylate as a ring-opening reagent can enhance the hydrophilicity of the adsorbent, improve the blood compatibility of the adsorbent, especially the adsorbent prepared by adding modified cyclodextrin with double bonds has significant positive effects on the adsorption of indoxyl sulfate, p-toluenesulfonic acid, beta2-microglobulin and parathyroid hormone; meanwhile, the 'amino solidification' and 'porogen' means have a positive promoting effect on the adsorption effect of the application;

[0126] (3) The adsorbent prepared by creatively adding glycidyl methacrylate as a ring-opening reagent has significant effects on the hemolysis and coagulation time ratio indexes of the biocompatibility of the adsorbent, and the 'amino solidification' technical feature has a significant effect on the coagulation time ratio index of the biocompatibility of the adsorbent, and has little effect on the hemolysis.

Claims

1. A method for preparing an adsorbent for removing protein-bound and medium- large molecule toxins, characterized by: The method comprises the following steps: (1) Preparation of polystyrene-based white balls: Mix styrene monomers, polyvinyl benzene, glycidyl methacrylate, 4,4'-bis(methacrylamido)-azobenzene, a porogen and an initiator as an oil phase, disperse, inorganic salt and pure water as an aqueous phase, mix the two phases, and perform suspension polymerization at a certain temperature and stirring rate to obtain polystyrene-based white balls, which are then cleaned, extracted and dried; (2) Amine solidification: The polystyrene-based white balls obtained in step (1) are subjected to amine treatment with an amine agent, and then cleaned to obtain amine polystyrene resin; (3) Film coating: The amine polystyrene resin is dissolved in a DMF solution, sodium carbonate is added, modified dextrin 6-OTs-β-CD is stirred and dissolved, N2 is introduced, and the reaction is performed under heating, followed by filtration, DMF-anhydrous ethanol precipitation and water washing to obtain an adsorbent. The porogen is one of toluene, xylene, chlorobenzene, 3-12 carbon atom alcohol, 5-12 carbon atom alkane, 200# gasoline or liquid paraffin, or a mixture of two to three kinds, and the addition amount of the porogen is 50%-300% of the total mass of the styrene monomers, polyvinyl benzene, glycidyl methacrylate and 4,4'-bis(methacrylamido)-azobenzene. Preparation method of modified dextrin 6-OTs-β-CD: After adding cyclodextrin β-CD into water, sodium hydroxide is slowly added dropwise into the flask, p-toluenesulfonyl chloride dissolved in acetonitrile is slowly added dropwise into the reaction solution under ice water bath conditions, the pH is adjusted after the reaction, and the product is extracted, recrystallized twice and vacuum dried to obtain modified cyclodextrin 6-OTs-β-CD. The amine agent comprises at least one of ethylenediamine, hexamethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine and polyethyleneimine.

2. The method of claim 1, wherein The styrene monomers are one or more of styrene, methylstyrene, ethylstyrene or 4-vinylbiphenyl; the polyvinyl benzene is one or more of divinylbenzene, divinyl ethyl benzene and triallyl isocyanurate.

3. The method of claim 1, wherein The initiator is one or more of azobisisobutyronitrile, azobisisoheptyl nitrile, benzoyl peroxide, tert-butyl peroxy-2-ethylhexanoate, tert-amyl peroxy-2-ethylhexanoate, dodecanoyl peroxide or alkyl hydroperoxide. The amount of the initiator is 0.5%-5% of the total mass of the styrene monomers, polyvinyl benzene, glycidyl methacrylate and 4,4'-bis(methacrylamido)-azobenzene.

4. The method of claim 1, wherein The dispersant is a water-soluble polymer, and the content is 0.1%-8% of the mass of the aqueous phase. The water-soluble polymer is one or two of polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), gelatin, polyethylene glycol, carboxymethyl cellulose or hydroxyethyl cellulose.

5. The method of claim 1, wherein In step (1), the mass ratio of the oil phase to the aqueous phase is 1:1-1:5; the reaction time is 10-20h; and the reaction temperature is 55-90℃.

6. The method of claim 1, wherein In step (1), the preparation method of 4,4'-bis(methacrylamido)-azobenzene comprises the following steps: To pyridine was added a solution of 4,4'-diaminoazobenzene and stirred at room temperature, methacryloyl chloride was gradually added, after complete addition the reaction mixture was heated, after cooling the mixture was poured into ice, acidified with hydrochloric acid, filtered, washed with saturated sodium bicarbonate solution and then with water, the crude product was recrystallized from ethanol.

7. An adsorbent for removing protein-bound and medium to large molecular weight toxins, prepared according to the method of any one of claims 1 to 6.

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